Relationship between Apolipoprotein E Genotype and Lipoprotein Profile in Patients with Coronary Heart Disease

(1) Background: Apolipoprotein E(ApoE) plays a critical role in lipid transport. The specific allele of APOE being expressed is associated with the development of coronary heart disease (CHD), however the specific mechanisms by which ApoE drives disease are unclear. In this study, we investigated the relationship between APOE allele, lipoprotein metabolome, and CHD severity to provide evidence for the efficacy of clinical cholesterol-lowering therapy; (2) Methods: Blood samples were collected from 360 patients with CHD that were actively being treated with statins. The lipoprotein profile, including particle numbers, particle size, and lipoprotein composition concentrates, was measured by nuclear magnetic resonance (NMR) spectroscopy. The severity of CHD was determined by quantifying coronary angiography results using the Gensini scoring system; (3) Results: We found there was no significant difference in low-density lipoprotein cholesterol (LDL-C) levels among ε2+ (ε2 allele carriers, consisting of ε2/ε2 and ε2/ε3 genotypes), ε3 (consisting of ε3/ε3 and ε2/ε4 genotypes), and ε4+ (ε4 allele carriers, consisting of ε3/ε4 and ε4/ε4 genotypes) participants receiving statin treatment. Compared with the ε3 group, patients with the ε2+ genotype showed lower concentrations of total low-density lipoprotein (LDL), small-LDL, and middle-LDL particles, as well as a larger LDL size, higher very low-density lipoprotein (VLDL) composition concentrates, and higher intermediate density lipoprotein (IDL) composition concentrates. The ε4+ group showed higher concentrations of total LDL, small LDL particles, and LDL compositions with smaller LDL size. The higher level of small LDL concentration was associated with a high Gensini score (B = 0.058, p = 0.024). Compared with the ε3 group, the risk of increased branch lesions in the ε2+ group was lower (OR = 0.416, p = 0.027); (4) Conclusions: The specific allele of APOE being expressed can affect the severity of CHD by altering components of the lipoprotein profile, such as the concentration of small LDL and LDL size.     

Particle size measurement of lipoprotein fractions using diffusion-ordered NMR spectroscopy

The sizes of certain types of lipoprotein particles have been associated with an increased risk of cardiovascular disease. However, there is currently no gold standard technique for the determination of this parameter. Here, we propose an analytical procedure to measure lipoprotein particles sizes using diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY). The method was tested on six lipoprotein fractions, VLDL, IDL, LDL₁, LDL₂, HDL₂, and HDL₃, which were obtained by sequential ultracentrifugation from four patients. We performed a pulsed-field gradient experiment on each fraction to obtain a mean diffusion coefficient, and then determined the apparent hydrodynamic radius using the Stokes–Einstein equation. To validate the hydrodynamic radii obtained, the particle size distribution of these lipoprotein fractions was also measured using transmission electron microscopy (TEM). The standard errors of duplicate measurements of diffusion coefficient ranged from 0.5% to 1.3%, confirming the repeatability of the technique. The coefficient of determination between the hydrodynamic radii and the TEM-derived mean particle size was r ² = 0.96, and the agreement between the two techniques was 85%. Thus, DOSY experiments have proved to be accurate and reliable for estimating lipoprotein particle sizes.     

Comparison of gel permeation chromatography, density gradient ultracentrifugation and precipitation methods for quantitation of very-low-, low- and high-density lipoprotein cholesterol.

Human VLDL, LDL and HDL (very-low-, low-, and high-density lipoproteins) were isolated from plasma by gel permeation chromatography with one pre-ultracentrifugation step. The column effluent was monitored at 280 nm. The cholesterol content of the fractions correlated well with fractions from sequential ultracentrifugation (VLDL, r = 0.839; LDL, r = 0.924; HDL, r = 0.766) or precipitation (LDL, r = 0.975; HDL, r = 0.972) methods. The average triglyceride, phospholipid and protein compositions of the separated lipoprotein fractions were close to those of the ultracentrifugally isolated fractions reported previously. Apolipoproteins A1 and B were determined from fractions to confirm the right distribution between different lipoproteins.     

人血清脂蛋白与胆固醇修饰的葡聚糖的相互作用

采用浊度法和高效液相色谱法研究了人血清低密度脂蛋白（LDL）和极低密度脂蛋白（VLDL）与胆固醇修饰的葡聚糖（CHD）的相互作用。浊度法研究结果表明，CHD与VLDL混合溶液的浊度大于CHD与LDL混俣溶液的浊度。CHD浓度和CHD链上胆固醇含量都对溶液的浊度产生影响。当CHD浓度为0．3mg/mL，CHD链上胆固醇含量为4个／100个糖环时，混合溶液的浊度可达到最大值，表明LDL或VLDL要的程度最大。Ca^2 对CHD与LDL或VLDL的相互作用没有影响。高效液相色谱的研究结果表明，CHD与LDL的结合使得复合物分子量增加；CHD分子量增大，复合物分子量也增加；当葡聚糖上胆固醇含量为4．0时，复合物分子量最大。实验结果表明，CHD能引起LDL或VLDL的自聚集。     

Determination of fatty acid compositions and cholesterol levels of some freshwater fish living in Porsuk Dam,Turkey

We determined the oil content, fatty acid composition, and cholesterol content of common carp (Cyprinus carpio), crucian carp (Carassius carassius), chub (Leusiscus cephalus), and tench (Tinca tinca) by GLC. The saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) levels were found to be 36.49%, 31.92%, 31.59% in common carp; 32.92%, 32.21%, and 34.87% in crucian carp; 36.19%, 32.91%, and 30.90% in chub; and 32.86%, 30.77%, and 36.37% in tench, respectively. The cholesterol (mg/100 g oil) levels of common carp, crucian carp, chub, and tench were determined by GLC methods as 119 ± 2.64 mg, 170.37 ± 2.36 mg, 94.68 ± 3.13 mg, and 179.84 ± 6.75 mg, respectively. Thus, the cholesterol contents of the analyzed freshwater fish species were low but their PUFA contents and nutritional values were high. Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 15–17, January–February, 2009.     

Identification and analysis of stereoselective drug interactions with low-density lipoprotein by high-performance affinity chromatography

Columns containing immobilized low-density lipoprotein (LDL) were prepared for the analysis of drug interactions with this agent by high-performance affinity chromatography (HPAC). R/S-Propranolol was used as a model drug for this study. The LDL columns gave reproducible binding to propranolol over 60 h of continuous use in the presence of pH 7.4 0.067 M potassium phosphate buffer. Experiments conducted with this type of column through frontal analysis indicated that two types of interactions were occurring between R-propranolol and LDL, while only a single type of interaction was observed between S-propranolol and LDL. The first type of interaction, which was seen for both enantiomers, involved non-saturable binding; this interaction had an overall affinity (nK _a) of 1.9 (±0.1) × 10⁵ M⁻¹ for R-propranolol and 2.7 (±0.2) × 10⁵ M⁻¹ for S-propranolol at 37 °C. The second type of interaction was observed only for R-propranolol and involved saturable binding that had an association equilibrium constant (K _a) of 5.2 (±2.3) × 10⁵ M⁻¹ at 37 °C. Similar differences in binding behavior were found for the two enantiomers at 20 °C and 27 °C. This is the first known example of stereoselective binding of drugs by LDL or other lipoproteins. This work also illustrates the ability of HPAC to be used as a tool for characterizing mixed-mode interactions that involve LDL and related binding agents.     

Simultaneous Determination of High-Density Lipoprotein,Very Low-Density Lipoprotein and Low-Density Lipoprotein Subclass in Human Serum by Microchip CE

Lipoproteins, especially high-density lipoproteins (HDL), very low-density lipoproteins (VLDL) and small, dense low-density lipoprotein (sdLDL), are believed to play an important role in the development of atherosclerosis. In this work, a simple, selective and sensitive method for the simultaneous monitoring of these lipoproteins in human serum using microchip capillary electrophoresis was developed. Gold nanoparticles were used as an additive to the running buffer to obtain the absolute separation of the lipoproteins. Under optimised conditions, the linear ranges of large buoyant low-density lipoproteins, sdLDL, VLDL and HDL were 10–800, 10–800, 40–1,000 and 20–800 μg L⁻¹, and their limits of detection were 5, 5, 15 and 8 μg L⁻¹, respectively. The intraassay and interassay relative standard deviation of lipoprotein peak areas were in the range of 3.8–7.4%. For practical application, variations in the serum lipoprotein of coronary heart disease patients were monitored by microchip-based CE. The results showed that the method was applicable for routine clinical use and allowed the rapid detection of different lipoprotein classes as well as their subclasses, thus greatly improving the analysis of atherosclerotic risk factors.

     

Simultaneous Determination of High-Density Lipoprotein, Very Low-Density Lipoprotein and Low-Density Lipoprotein Subclass in Human Serum by Microchip CE

Lipoproteins, especially high-density lipoproteins (HDL), very low-density lipoproteins (VLDL) and small, dense low-density lipoprotein (sdLDL), are believed to play an important role in the development of atherosclerosis. In this work, a simple, selective and sensitive method for the simultaneous monitoring of these lipoproteins in human serum using microchip capillary electrophoresis was developed. Gold nanoparticles were used as an additive to the running buffer to obtain the absolute separation of the lipoproteins. Under optimised conditions, the linear ranges of large buoyant low-density lipoproteins, sdLDL, VLDL and HDL were 10?C800, 10?C800, 40?C1,000 and 20?C800 ??g L^?1, and their limits of detection were 5, 5, 15 and 8 ??g L^?1, respectively. The intraassay and interassay relative standard deviation of lipoprotein peak areas were in the range of 3.8?C7.4%. For practical application, variations in the serum lipoprotein of coronary heart disease patients were monitored by microchip-based CE. The results showed that the method was applicable for routine clinical use and allowed the rapid detection of different lipoprotein classes as well as their subclasses, thus greatly improving the analysis of atherosclerotic risk factors.     

Lipoproteins modulate growth and differentiation of cultured human epidermal keratinocytes

The effects of various lipoproteins on the growth and the differentiation of cultured normal human keratinocytes were investigated. Primary cultures of human epidermal keratinocytes were obtained from neonatal foreskin, and then added with lipoproteins, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Cell growth potential was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. VLDL and LDL enhanced keratinocytes growth and LDL receptor expression at the plasma membrane level. These effects were more remarkably observed in cells cultured with VLDL than in cells cultured with LDL. Apolipoprotein E (ApoE) was highly increased in VLDL treated cells. These results suggest that VLDL binds with high affinity to cell surface receptors and induces cell proliferation.     

Separation of molecular tracers sorbed onto atmospheric particulate matter by flash chromatography

In to order increase sensitivity and to reduce the background induced by matrix effects, a method was developed that uses flash chromatography to separate various compounds present in atmospheric aerosol samples prior to their analysis with different analytical techniques (GC–MS, GC–FID, HPLC). For this purpose, flash chromatography using a 4 g silica gel column crossed by eluent at a flow rate of 20 mL min⁻¹ was used. An eluent with enhanced polarity is needed to separate nonpolar (linear and branched alkanes), semipolar (PAH, nitro-PAH and cholesterol) and polar (methoxyphenols, alkanoic acids, and levoglucosan) compounds. Three combinations of solvents were used: hexane for the nonpolar fraction (F1), toluene/hexane for the semipolar fraction (F2) and dimethylformamide for the polar fraction (F3). The use of different eluents for each fraction allows separation of the sample to be accomplished with good repeatability and satisfying yields [85 ± 5% for F1, 81 ± 8% (PAHs), 89 ± 6% (nitro-PAHs) and 74 ± 7% (cholesterol) for F2 and 79 ± 7% (n-alkanoic acids), 40 ± 11% (methoxyphenols) and 77 ± 6% (levoglucosan) for F3]. The methoxyphenol yields were low due to losses during the concentration/evaporation step. This method was then applied to analyse the organic composition of particles collected at an urban site in Strasbourg (France).     

Über die Wirkung von Albumin auf die elektrophoretische Wanderungsgeschwindigkeit der menschlichen Serumlipoproteine auf Agarose-Gel

The electrophoretic mobility of the VLDL, LDL and HDL fractions increases on agarose gel after Sepharose 2 B gel filtration. This effect is broken of by albumin. The possibility of spontaneous hydrolysis of the triglycerides of the lipoproteins with a successive increase of the free fatty acid content and electric charge is discussed. The albumin effect is based on the binding of these free fatty acids.     

Fractionation of human serum lipoproteins and simultaneous enzymatic determination of cholesterol and triglycerides

A method based on Asymmetric Flow Field-Flow Fractionation (AF4) was developed to separate different types of lipoproteins from human serum. The emphasis in the method optimization was on the possibilities to characterize the largest lipoprotein fractions (LDL and VLDL), which is usually not possible with the size-exclusion chromatography methods applied in routine analysis. Different channel geometries and flow programs were tested and compared. The use of a short fractionation channel was shown to give less sample dilution at the same fractionation power compared to a conventional, long channel. Different size selectivities were obtained with an exponential decay and a linear cross flow program. The ratio of the UV absorption signal to the light scattering signal was used to validate the relation between retention time and size of the fractionated particles.An experimental setup was developed for the simultaneous determination of the cholesterol and triglycerides distribution over the lipoprotein fractions, based on enzymatic reactions followed by UV detection at 500 nm. Coiled and knitted PTFE tubing reactors were compared. An improved peak sharpness and sensitivity were observed with the knitted tubing reactor. After optimization of the experimental conditions a satisfactory linearity and precision (2-3% rsd for cholesterol and 5-6% rsd for triglycerides) were obtained. Finally, serum samples, a pooled sample from healthy volunteers and samples of sepsis patients, were analyzed with the method developed. Lipoprotein fractionation and cholesterol and triglyceride distributions could be correlated with the clinical background of the samples.     

Production of Lipid and Fatty Acids during Growth of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> on Hydrocarbon Substrates

An Aspergillus terreus, isolated from oil contaminated soil, could degrade a wide range of petroleum hydrocarbons including the immediate oxidation products of hydrocarbons, like alkanols and alkanals. Among all the linear chain carbon substrates, highest growth of 39.1 ± 3.8 g l⁻¹ (wet weight) was observed when n-hexadecane was used as the sole source of carbon. The growth of the fungus on this highly hydrophobic substrate was associated with the morphological change of the hyphae and increase production of lipid in the cells. The lipid production in the hydrocarbon (n-hexadecane) grown cells was sevenfold higher than the corresponding glucose grown cells. The fatty acid profile of the lipid content formed in the hydrocarbon grown cells was significantly different from the glucose grown cells and was composed of fatty acids with chain length C₁₄ to C₃₃ as revealed from the liquid chromatography electrospray ionization mass spectrometry analyses. Among the ranges, the fatty acids with chain lengths C₁₄ to C₁₈ were predominant in the profile. Considering the fatty acid profile and the high level of lipid production, this A. terreus mediated production of lipid is envisaged to have potential application in the oleochemical industries including the production of biodiesel.     

Secreted phospholipase A<Subscript>2</Subscript>, lipoprotein hydrolysis,and atherosclerosis: integration with lipidomics

Phospholipase A₂ (PLA₂) is a group of enzymes that hydrolyze the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. Of many PLA₂s or related enzymes identified to date, secreted PLA₂s (sPLA₂s) comprise the largest family that contains 10 catalytically active isozymes. Besides arachidonic acid released from cellular membranes for eicosanoid synthesis, several if not all sPLA₂s have recently been implicated in hydrolysis of phospholipids in lipoprotein particles. The sPLA₂-processed low-density lipoprotein (LDL) particles contain a large amount of lysophospholipids and exhibit the property of “small-dense” or “modified” LDL, which facilitates foam cell formation from macrophages. Transgenic overexpression of these sPLA₂s leads to development of atherosclerosis in mice. More importantly, genetic deletion or pharmacological inhibition of particular sPLA₂s significantly attenuates atherosclerosis and aneurysm. In this article, we will give an overview of current understanding of the role of sPLA₂s in atherosclerosis, with recent lipidomics data showing the action of a subset of sPLA₂s on lipoprotein phospholipids.     

Distribution of Arsenic Species in Different Leaf Fractions – An Evaluation of the Biochemical Deposition of Arsenic in Plant Cells

This paper describes an approach to the determination of arsenic species bonding with proteins or low-molecular peptides by separation of leaf proteins and protein precursors into three fractions and analysis of arsenic species associated to these fractions. Plants irrigated with arsenite contained not only arsenite but also arsenate and dimethylarsinate. In plants treated with arsenate, the major component was arsenite in the water-soluble fraction containing soluble protein and non-protein (F II) and in the acid-soluble non-protein fraction (F IV). Concentrations of 43 mg kg⁻¹ (As(V)-treatment) and 18 mg kg⁻¹ (As(III)-treatment) could be analyzed in the water-insoluble structure protein fraction F I (56 ± 15% of the total mass). Based on the concentration of arsenic species in all fractions, conclusions are drawn over the fixation of arsenic in the fraction of insoluble structure proteins, in the fraction of soluble cytosolic proteins as well as the fraction of amino acids.     

Ellipsometry Studies of Lipoprotein Adsorption

The adsorption of a number of lipoproteins, i.e., low-density lipoprotein (LDL), oxidized LDL (oxLDL), high-density lipoprotein (HDL), and lipoprotein (a), at silica and methylated silica as well as at the latter surface modified through adsorption of proteoheparan sulfate, was investigated with in situ ellipsometry at close to physiological conditions. It was found that LDL, oxLDL, HDL, and lipoprotein (a) all adsorbed more extensively at silica than at methylated silica. Upon exposure of the methylated silica surface to proteoheparan sulfate, this proteoglycan adsorbs through its hydrophobic moiety, thereby forming a layer similar to that in the biological system, with the polysaccharide chains forming brushes oriented toward the aqueous solution. Analogous to the biological system, both lipoprotein (a) and LDL were found to deposit at such surfaces, the latter particularly in the simultaneous presence of Ca(2+). After HDL pre-exposure, however, no LDL deposition was observed, even at high LDL and Ca(2+) concentrations. These findings correlate well with those obtained from clinical investigations on risk factors for atherosclerosis. Copyright 2000 Academic Press.     

Myocardial infarction patients show altered lipoprotein properties and functions when compared with stable angina pectoris patients

Several parameters and risk factors were compared between Korean male myocardial infarction (MI) patients (n = 10) and angina pectoris (AP) patients (n = 17) to search unique biomarkers for myocardial infarction (MI) in lipoprotein level. Individual serum and lipoprotein fractions (VLDL, LDL, HDL₂, HDL₃) were isolated and analyzed by lipid and protein determination and enzyme assay. The MI group was found to have a 25 and 30% higher serum cholesterol and triacylglycerol (TG) level than the AP group, respectively, however, their body mass index (BMI), LDL-cholesterol (C), HDL-C, and glucose levels fell within the normal range. MI patients were found to have an approximately two-fold higher level of serum IL-6 and an 18% lower serum apoA-I level than that of the AP group. LDL and HDL₂ fraction of the MI group were more enriched with TG than those of AP group. The increased TG was correlated well with the increased level of apoC-III in the same fraction. Cholesteryl ester transfer protein (CETP) activity and protein level were greatly increased in MI patients in the LDL and HDL₃ fractions. MI patients showed more severely oxidized LDL fraction than patients in the AP group, as well as the weakest antioxidant ability of serum. Conclusively, MI patients were found to have unique serum and lipoprotein characteristics including increased IL-6 and TG in serum, with CETP and apoC-III in the LDL and HDL fractions, as well as severely impaired antioxidant ability of HDL.     

Electrophoretic behavior and partial characterization of disease-associated serum forms of gamma-glutamyltransferase

We have recently devised an improved procedure for the rapid electrophoretic separation of multiple forms of serum gamma-glutamyltransferase (GGT). This procedure is based on the separation on cellulose acetate strips, usually employed for lipoprotein electrophoresis, followed by visualization with a fluorescent reagent. The method is highly sensitive and the fractions are more clearly resolved than with other procedures. Reference intervals have been evaluated in the sera from 142 healthy subjects and the patterns (two GGT forms comigrating with alpha 1 and alpha 2-globulin) are reproducible. In 150 sera from patients with various hepatobiliary diseases (including neoplasias), acute pancreatitis and non liver-involving neoplasias, we observed some disease-specific GGT forms: an albumin comigrating enzyme (Alb-GGT) specific of liver neoplasia; a gamma-globulin comigrating GGT (gamma-GGT) and a nonmigrating isoform (dep-GGT) both specifically associated to extrahepatic jaundice. Multiple lipoprotein fraction precipitation showed that beta-, gamma- and dep-GGT are complexes between GGT and low density lipoprotein and very low density lipoproteins (LDL + VLDL), and that some of the alpha 1-GGT from cirrhotic patients is a complex between GGT and high density lipoprotein (HDL). GGT fractions from normal subjects and Alb-GGT from patients with liver neoplasia do not appear to be complexed with lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the oxidative status of virgin olive oils with different phenolic content by direct infusion atmospheric pressure chemical ionization mass spectrometry

Atmospheric pressure chemical ionization mass spectrometry was used to predict the oxidative status of virgin olive oils (VOO) during their storage. VOO samples, with and without phenolic compounds, were stored in the dark at 60 °C up to 7 weeks. The VOO samples were diluted in an alkaline propanol/methanol mixture and directly infused into an ion-trap mass spectrometer. The abundances of the [M−H]⁻ peaks of free fatty acids, oxidized fatty acids, tocopherols and phenolic compounds, jointly with their oxidized forms, were measured and used as predictors. Two linear discriminant analysis (LDA) models were constructed in order to classify samples according to their oxidative levels. The first model was constructed using both VOO samples (with and without phenols), considering as predictors only fatty acids and their oxidized products. The second LDA model was constructed with the VOO sample with phenolic compounds considering as predictors all the peaks measured. In both models, the samples divided in the eight different storage times were correctly classified (100%) by leave-one-out cross-validation with an excellent resolution among all the category pairs (for the first model Wilks’ lambda, λ _w = 0.229 and for the second λ _w = 0.928). This method is a very fast tool for on-line monitoring of VOO oxidation status.